Mycobacterium tuberculosis remains one of the most devastating human infectious diseases, causing two million deaths annually and latently infecting a third of the world's population. As an intracellular pathogen adapted to long-term survival, M. tuberculosis has evolved mechanisms to manipulate host events that rely on dynamic membrane processes such as phagosome maturation, phagolysome fusion and autophagy. Other pathogenic bacteria achieve similar effects by secreting protein virulence factors (called effector proteins or effectors) that associate with host membranes to facilitat their activities. To date, few such effectors have been identified in M. tuberculosis and expanding this knowledge base may provide additional avenues for the development of new drugs. Because eukaryotic cellular membranes are the major organizational centers of the cell, we hypothesize that membranes are targeted by mycobacterial effector proteins. In preliminary experiments, we tested putative secreted mycobacterial effectors for their ability to bind host membranes using a high-throughput screening assay in the model eukaryotic organism, Saccharomyces cerevisiae (yeast). Of the 40 genes screened to date, 5 (12.5%) interact with membranes, and we have demonstrated that in a human cell line several of the genes associate with the major protein synthesis machinery center of the cell, the endoplasmic reticulum. The aims of this proposal are to fundamentally understand how M. tuberculosis is able to use membrane targeting to manipulate the host. We propose to first identify a complete complement of membrane binding proteins secreted by M. tuberculosis by screening a total of 400 genes using our high- throughput cloning and expression system. Characterization will further include cell biologic assays to determine where each protein associates within eukaryotic cells and demonstration of direct secretion of mycobacterial effectors into host cells during infection. We will select several hits for further in-depth characterization including in vitro assays of membran association and identification of host protein targets. Lastly, we will use the knowledge gained to test the hypothesis that regulation of membrane and organelle dynamics by M. tuberculosis effectors is essential for mycobacterial survival within human macrophages. The proposed work will extend the current knowledge on M. tuberculosis's ability to manipulate host membrane dynamics and reveal novel microbial survival strategies.